1. LECTURE 18: TRANSPOSABLE ELEMENTS gchapter 13 gexam 2 & grades ggeneral ideas gdiscovery in maise gprokaryotes geukaryotes gdynamic & plentiful ghost regulation

2. The Biology Graduate Student Association Presents A forum to present current and prospective research projects in the field of Biology Barrick Museum Auditorium Saturday, November 18th 8:00 am to 2:30 pm

3. Fyou need a piece of paper and a pen or pencil... Fwrite your name and student number at the top... Fgive brief answers for the questions below... Q1: Transposable elements were first discovered in _________. Q2: Name the 2 classes of eukaryotic transposons and describe some of their defining features. LECTURE 21 TRANSPOSABLE ELEMENTS QUIZ #5

4. TRANSPOSABLE ELEMENT QUESTIONS g why were they 1 st discovered in corn but 1 st isolated from E. coli ? g how do they confer antibiotic resistance in bacteria? g why the classification as DNA or RNA transposons? g what do autonomous and non-autonomous mean? g what are the implications for the human genome with  50% of it derived from transposable elements

5. GENERAL IDEAS g Barbara McClintock 1920s  1980s (nobel in 1984) g mostly ignored for decades g  50% of human genome g 2 types class 1. retrotransposons g transcribe class 2. DNA elements g excise

6. DISCOVERY IN MAISE g Barbara McClintock 1920s  1980s (Nobel in 1983) g “Indian” corn, 10 chromosome pairs g Dissociation ( Ds ) factor on # 9 commonly broken g Activator ( Ac ) unlinked factor required for breaks g could not map to constant place g lots of bizarre kernel phenotypes, not ~ parents

7. DISCOVERY IN MAISE g mosaicism through the activity of Ds movement g during mitosis, results in patchy tissue g chromosome breakage & loss g all linked genes affected

8. DISCOVERY IN MAISE g mosaicism through the activity of Ds movement g during mitosis, results in patchy tissue g unstable insertions & excission g single gene affected only, e.g. C gene

9. DISCOVERY IN MAISE g mosaicism through the activity of Ds movement g during mitosis, results in patchy tissue g size of mutant sector ~ time of event

10. DISCOVERY IN MAISE g autonomous & nonautonomous elements g C gene, allele called c-mutable(Ds) or c-m(Ds) g Ds stable without Ac g Ds excision is dependent on Ac g Ds is nonautonomous g C gene, allele called c-mutable(Ac) or c-m(Ac) g Ac always unstable g Ac is autonomous g Ac can transform  Ds g Ds = Ac mutant

11. DISCOVERY IN MAISE g autonomous & nonautonomous elements g Ds & Ac are members of a transposable element family g many other families discovered in maize g autonomous elements encode information necessary for the transposition of themselves and nonautonomous members of their family

12. PROKARYOTES g molecular biology of transposable elements first characterized in bacteria g insertion sequence (IS) elements g transposable g can block gene & operon function (polar) g e.g., gal operon in E. coli

13. PROKARYOTES g are the gal mutants all the same?... NO g several different IS sequences g inserted in different places g all encode transposase enzyme g multiple copies,  can recombine F factor

14. PROKARYOTES g 2 types of bacterial transposons 1.composite: genes ( e.g., drug resistance) sandwiched between inverted IS sequences... g in this case, these are called inverted repeat (IR) sequences

15. PROKARYOTES g 2 types of bacterial transposons 2.simple: genes sandwiched between IR sequences g genes include transposase g IR sequences are short (<50 bp) & do not encode transposase

16. PROKARYOTES g transposons can tranpose (jump) to & from plasmids & chromosomes g implications for drug resistance

17. PROKARYOTES g basic mechanism of tranposition g transposase makes staggered cuts in host DNA g element inserts g host DNA repair fills in gaps g in this example, it generates 5-bp direct repeats on either side g called target-site duplications

18. PROKARYOTES g 2 types (at least) of transposition mechanisms 1.replicative: copy remains in original site 2.conservative (nonreplicative): excision only

19. PROKARYOTES g 2 types (at least) of transposition mechanisms 1.replicative: copy remains in original site g recombination event

20. EUKARYOTES g 2 types of eukaryotic transposons class 1. retrotransposons g transcription mechanism class 2. DNA transposons g excision mechanism

21. EUKARYOTES class 1. retrotransposons g resemble single stranded RNA retroviruses g copied into DNA using reverse transcriptase g inserts into host g transcribes new viral genome & proteins  new viral particles g called provirus when integrated

22. EUKARYOTES class 1. retrotransposons g resemble single stranded RNA retroviruses g similar structure & gene content g flanked by long terminal repeat sequences (LTRs) 100s of bp long g these are also called LTR-retrotransposons

23. EUKARYOTES class 1. retrotransposons g gag : maturation of RNA genome g pol : reverse transcriptase g env : protein coat (viral gene only)

24. EUKARYOTES class 1. retrotransposons g Ty elements in yeast g copia -like elements in Drosophila g 10-100 positions in genome g cause known mutations, e.g., w a w + w a w 1118

25. EUKARYOTES class 2. DNA transposons g mechanisms similar to those in bacteria g Drosophila P -elements 1 st characterized g discovered ~ hybrid dysgenesis

26. EUKARYOTES class 2. DNA transposons g mechanisms similar to those in bacteria g Drosophila P -elements first characterized g discovered ~ hybrid dysgenesis g P -strains have 30 - 50 P -element copies / genome g 2.9 kb wild type element, 31 bp inverted repeats g defective elements are smaller g tranposase gene has 3 introns + 4 exons

27. EUKARYOTES class 2. DNA transposons g hybrid dysgenesis mechanism in Drosophila

28. EUKARYOTES class 2. DNA transposons g action of Ac element in maise

29. EUKARYOTES g DNA transposons, gene discovery & manipulation g controlled use of engineered P -elements g 2 element system: 1.  2-3: g transposase source g disrupted terminal IR sequences g stable (immobilized) 2.bullet: g deleted transposase gene g inserted genes of interest ( e.g. markers) g mobilized only in combination with #1

30. EUKARYOTES g DNA transposons, gene discovery & manipulation g controlled use of engineered P -elements g genes of interest inserted in bullet g gene transfer...  g re-mobilization

31. EUKARYOTES g DNA transposons, gene discovery & manipulation g controlled use of engineered P -elements g insertional mutagenesis g provide transposase for 1 generation g cross away & screen for new mutants g use P -element sequence to probe for gene = transposon tagging g enhancer trap mutagenesis g finds functional regulatory sequences g GAL4 system (binary, 2 bullets) g tool for gene manipulation

32. x  P- ELEMENTS & YEAST GAL4 SYSTEM w – E X / w– w– / GAL4 w + GFP w +

33. / GAL4 w + w – E X / w– w– GFP w + green fluorescent protein P- ELEMENTS & YEAST GAL4 SYSTEM

34. CYTOPLASMIC SIGNAL NUCLEAR SIGNAL P- ELEMENTS & YEAST GAL4 SYSTEM MUSHROOM BODY KENYAN CELL

35. DYNAMIC & PLENTIFUL g DNA content of organism  C-value g lack of correlation with biological complexity  C-value paradox g DNA repeat sequences make up large fraction of eukaryotic genomes g genome size correlates with amount of DNA derived from transposable elements g e.g., ~ half of the human genome is derived from transposable elements

36. DYNAMIC & PLENTIFUL g human genome g long interspersed nuclear elements (LINES) g autonomous, retrotranspose, no LTRs g short interspersed nuclear elements (SINES) g nonautonomous, ~ lines w/o rev. transcriptase g Alu element ~ 10% of genome

37. DYNAMIC & PLENTIFUL g human genome g ~ 20  as much DNA derive from transposable elements as protein-encoding DNA g intron insertions remain only  spiced out g presumably initially also in exons  mutations & negative selection g typical pattern in humans...

38. DYNAMIC & PLENTIFUL g human genome g class 1 transposons (LINES, SINES) cause some hereditary diseases in humans, e.g., g hemophilia A g neurofibromatosis g breast cancer g class 2 transposons (DNA) g low mutation rate (0.2 % or 1 in 500 known)

39. DYNAMIC & PLENTIFUL g plants ( e.g. grasses) g synteny: similar gene content & organization g vastly different genome sizes due to transposons g safe havens: strategy of insertion in other transposons, minimize negative effect on host transposons genes

40. DYNAMIC & PLENTIFUL g yeast g small genome, 70% exons g Ty LTR-retrotransposons g targeted insertions to benign sites g encoded integration enzyme

41. DYNAMIC & PLENTIFUL g Drosophila g telomeres are transposable elements! g HeT-A & TART non-LTR retrotransposons (LINES) g telomerase is a reverse transcriptase g RNA template for telomere DNA synthesis

42. HOST REGULATION g Ac activity reversible g lost of activity reappeared in later generations g epimutations: changes in chromatin structure

43. HOST REGULATION g transgene silencing g cosuppression: transformed gene & endogenous homologous genes both silenced g unknown defense mechanism?

44. SPEND SOME TIME WITH... g key questions revisited (p.446-447) g summary (p.447) g terminology (p.447-8) g unsolved problems (p.449)... g 2, 3, 5, 7, 11